Split-GFP tagging and live imaging of hair cell proteins

在过去的几十年里，毛细胞领域已经对毛细胞的内部运作有了很大的了解， 调节我们的听觉和平衡感的感觉感受器。许多因子的分子同一性 对毛细胞发育和功能的重要性，以及它们对听力损失的意义已经阐明。 尽管取得了这些进展，毛细胞的研究仍面临许多挑战。特别是活细胞成像， 蛋白质的追踪是细胞生物学研究的重要支柱，一直特别困难，主要是由于有限的 是指毛细胞的分离和培养。考虑到毛细胞功能的本质是微观的， 纳米运动，蛋白质的动态特性的研究是至关重要的一个完整的 了解毛细胞的功能。因此，本提案的目标是开发一种工具， 活毛细胞中的蛋白质运动更容易获得。 追踪蛋白质运动的黄金标准技术是标记蛋白质的活细胞成像。传统上， 这是通过表达与荧光蛋白融合的外源DNA构建体来实现的， 基因枪、电穿孔或病毒。转基因小鼠也已被采用。然而，这些方法， 可能被过表达假象和低转导/转染效率所混淆。敲入 (KI)将遗传编码的荧光标记插入内源基因位点已经变得更加容易处理， 现代基因组编辑方法，但大DNA片段的KI，如全长GFP编码序列， 仍然很困难，并有可能影响基因表达。 为了应对这些挑战，我们计划开发一种小鼠工具包， 蛋白质定位和活细胞成像。为此，我们采用了Split-GFP策略。在这两个- 在一个组分系统中，用一小部分GFP（“GFP 11”）对感兴趣的内源基因进行遗传标记。 共表达GFP的剩余（非荧光）部分（“GFP 1 -10”）重建GFP荧光， 允许通过荧光显微镜成像。由于GFP 11片段的小尺寸（48 bps），CRISPR- 介导的KI进入内源基因座是高效的。在初步研究中，我们证实了Split-GFP 这种方法忠实地再现了表皮板蛋白LMO 7的内源性定位，通过递送 AAV包装的GFP 1 -10进入Lmo 7-GFP 11 KI小鼠的毛细胞。为了规避AAV介导的递送，我们 提出产生表达GFP 1 -10片段的转基因小鼠系。创始人已经 获得并证实了生殖系传播（SA 1）。该系统将在实时成像上进行测试 增加难度的任务：在SA 2中，我们将研究LMO 7在毛细胞中的扩散动力学和运动。 在SA 3中，挑战该系统的灵敏度极限，我们将研究尖端连杆电机的动态特性 复杂的组件USH 1C的头发束。

Over the past few decades, the hair cell field has gained great insight into the inner workings of hair cells, the sensory receptors that mediate our sense of hearing and balance. The molecular identity of many factors important for hair cell development and function, and their significance for hearing loss, have been elucidated. Despite this progress, the study of hair cells presents many challenges. In particular live-cell imaging and tracking of proteins, important pillars of cell biology studies, have been especially difficult, mainly due to limited means to transfect and culture hair cells. Considering that the essence of hair cell function is micro- and nanoscale movement, investigations of the dynamic properties of proteins is crucial for a complete understanding of hair cell function. The goal of this proposal therefore is to develop a tool that makes imaging of protein movements in living hair cells more accessible. The gold standard technique for tracking protein movement is live-cell imaging of tagged proteins. Traditionally, this is achieved by expression of exogenous DNA constructs fused to fluorescent proteins, delivered to cells by gene gun, electroporation or viruses. Transgenic mice also have been employed. These approaches, however, are potentially confounded by overexpression artifacts and low transduction/transfection efficiency. Knock-in (KI) of genetically-encoded fluorescent tags into the endogenous gene loci has become more tractable through modern genome editing methods, but KI of large DNA segments, such as the full-length GFP coding sequence, remains difficult and has the potential to affect gene expression. To address these challenges, we plan to develop a mouse tool kit that streamlines fluorescent tagging of proteins for localization and live cell imaging. To this end, we adopted the Split-GFP strategy. In this two- component system, the endogenous gene of interest is genetically tagged with a small part of GFP (“GFP11”). Co-expressing the remaining (non-fluorescent) portion of GFP (“GFP1-10”) reconstitutes GFP fluorescence, allowing imaging by fluorescence microscopy. Due to the small size of the GFP11 fragment (48 bps), CRISPR- mediated KI into the endogenous loci is highly efficient. In preliminary studies, we confirmed that the Split-GFP approach faithfully recapitulates the endogenous localization of the cuticular plate protein LMO7, by delivering AAV-packaged GFP1-10 into the hair cells of Lmo7-GFP11 KI mice. To circumvent AAV-mediated delivery, we propose to generate a transgenic mouse line that expresses the GFP1-10 fragment. Founders have already been obtained and germline transmission was confirmed (SA1). This system will be tested on live-imaging tasks of increasing difficulty: In SA2, we will study the diffusion kinetics and movement of LMO7 in the hair cell. In SA3, challenging the sensitivity limits of this system, we will investigate the dynamics of the tip link motor complex component USH1C in the hair bundle.